JP5317056B2 - Identification and utilization of transporters responsible for chemical transmission of aspartic acid - Google Patents

Abstract

It is intended to identify a transporter in charge of aspartic acid transport and a gene encoding the transporter, and provide a screening method for an agent for regulating the activity of a transporter using an artificial membrane containing such a transporter. Also it is intended to provide a candidate substance of a preventive or therapeutic agent for a disease associated with aspartic acid metabolism by such a screening method. According to the invention, a transporter in charge of aspartic acid transport and a gene encoding the transporter were identified. Further, a screening method for an agent for regulating the activity of an aspartic acid transporter and a screening method capable of providing a candidate substance of a preventive or therapeutic agent for a disease associated with aspartic acid metabolism using such a transporter are provided.

Description

  The present invention relates to the field of novel aspartic acid transporters. Furthermore, the present invention relates to screening for drugs that modulate the transport activity of aspartic acid transporters, and screening for drugs for preventing / treating diseases associated with abnormal metabolism of aspartic acid.

Aspartic acid was originally identified as a chemotactic factor for bacteria and has a long history as a chemical transmitter. In the brains of mammals including humans, it has been found more than 30 years ago that when brain slices are stimulated, they are released together with glutamic acid and γ-aminobutyric acid (GABA). All amino acids have been considered to be neurotransmitters because they exhibit excitatory (or inhibitory) action on nerves. In order for these amino acids to function as chemical transmission, (1) they are first concentrated in synaptic vesicles present at the nerve terminals and then exocytosed in the synaptic cleft, (2) the released amino acids are receptors (3) After that, it is necessary to be absorbed by nerves or glial cells and disappear from the synaptic cleft. The last process is governed by the Na ⁺ driven transporter present in the plasma membrane. During these 30 years, the protein factors governing the above (1-3) in glutamic acid and γ-aminobutyric acid have been elucidated. That is, with respect to glutamic acid, three types of vesicularglutamate transporters VGLUT1, VGLUT2, and VGLUT3) (referred to as SLC17A6-8 in the classification as transporters) are present in synaptic vesicles and govern the concentration of glutamic acid in the vesicles. The released glutamate binds to various glutamate receptors, transmits a signal, and is collected by the plasma membrane glutamate transporter. Regarding chemical transfer of γ-aminobutyric acid, vesicular GABA transporter, GABA receptor and plasma membrane type GABA transporter are already known. On the other hand, the chemical transmission mechanism of aspartic acid was unknown. Aspartic acid is the same acidic amino acid as glutamic acid (which becomes an anion in vivo), but has one less carbon. Aspartic acid is particularly abundant in the hippocampus. In the hippocampal CA3 nerve endings, aspartic acid is contained in synaptic vesicles. Released by stimulation (Non-Patent Document 1: Zhouetal, 1995, Journal of Neurochemistry 64, 1556-1566). The released aspartic acid is thought to bind to the NMDA receptor and transmit a signal. In addition, extracellular aspartic acid is a good substrate for the protoplasmic glutamate transporter and is efficiently reabsorbed into the cell. Furthermore, the present inventors have reported that the pineal gland also concentrates aspartic acid and exocytoses (Non-patent Document 2: Yatsushiroetal, 1997 J Neurochem. 69, 340-347).

  Due to the characteristics of aspartic acid as a neurotransmitter shown above, a drug that regulates the function of aspartic acid transporter is useful for the prevention and treatment of diseases of nerve cells, for example, diseases related to CA3 nerve of hippocampus. It is expected that. Therefore, it is considered that the present invention makes it possible to provide a screening method that provides a candidate substance for a prophylactic / therapeutic agent for a neuronal disease, for example, a disease associated with the hippocampal CA3 nerve.

  Thus, identification of a gene encoding a protein responsible for aspartic acid transport has been desired.

  In order to demonstrate the chemical transfer mechanism of aspartate, it was necessary to show the existence of a transporter (virtual vesicular-type aspartate transporter, VaspT) that accumulates in synaptic vesicles. . However, since VGLUT does not use aspartic acid as a substrate (Non-patent Document 3: Moriyama Yand Yamamoto A, J Biochem (Tokyo). 135, 155-163), the mechanism for concentrating aspartic acid in synaptic vesicles is at least glutamic acid. It is thought that it is transported by a mechanism different from the transport mechanism, and the specific protein involved in the mechanism was unknown.

Prior art document information relating to the invention of this application includes the following.
Zhou et al, 1995, Journal of Neurochemistry 64, 1556-1566 Yatsushiro et al, 1997, J Neurochem. 69, 340-347 Moriyama Y and Yamamoto A, J Biochem (Tokyo) .135, 155-163

  It is an object of the present invention to identify a transporter responsible for aspartate transport and a gene encoding the transporter, and further to provide a screening method for a transporter activity regulator using an artificial membrane containing such a transporter. And Furthermore, it is also an object of the present invention to provide candidate substances for prophylactic / therapeutic agents for diseases involving aspartic acid metabolism by such screening methods.

  The present inventors have completed the present invention by identifying an aspartic acid transporter and analyzing its function by an original method described in the following Examples.

The present invention provides the following.
(Item 1) A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and that encodes a polypeptide having aspartic acid transport activity,
A nucleic acid comprising a sequence that is at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide having aspartate transport activity;
A nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, An artificial membrane comprising a polypeptide encoded by a nucleic acid selected from the group consisting of nucleic acids encoding a polypeptide having aspartate transport activity.
(Item 2) The artificial membrane according to item 1, which is a membrane vesicle.
(Item 3) The artificial membrane according to Item 1, which is a liposome.
(Item 4) The artificial membrane according to Item 1, further comprising a proton pump.
(Item 5) The artificial membrane according to item 4, wherein the proton pump is F _o F ₁ -ATPase.
(Item 6) The artificial membrane according to item 1, further comprising a membrane potential forming agent.
(Item 7) The artificial membrane according to item 6, wherein the membrane potential forming agent is valinomycin.
(Item 8) A screening method for an activity regulator of aspartate transport protein,
(A) providing the artificial membrane according to item 1;
(B) contacting the candidate drug with the artificial membrane;
(C) measuring the aspartic acid transport activity of the artificial membrane; and
(D) determining whether the candidate drug is an activity regulator of aspartate transport protein from the aspartate transport activity measured in step (c);
Including the method.
(Item 9) The method according to item 8, wherein the activity regulator is an inhibitor.
(Item 10) The method according to item 8, wherein the activity regulator is an activity promoter.
(Item 11) An activity regulator of aspartate transport protein obtained by the method according to Item 8.
(Item 12) The method according to item 8, wherein the artificial membrane of (a) further comprises a proton pump.
(Item 13) The proton pump is an _F o _F 1 -ATPase, The method of claim 12.
(Item 14) The method according to item 8, wherein the artificial membrane of (a) further contains a membrane potential forming agent.
(Item 15) The method according to item 14, wherein the membrane potential forming agent is valinomycin.
(Item 16) A composition for screening an activity regulator of aspartate transport protein, comprising the artificial membrane according to item 1.
(Item 17) The composition according to item 16, further comprising a proton pump.
18. The proton pump is an _F o _F 1 -ATPase, composition of claim 17.
(Item 19) The composition according to item 16, further comprising a membrane potential forming agent.
(Item 20) The composition according to item 19, wherein the membrane potential forming agent is valinomycin.
(Item 21) a) A polypeptide comprising:
A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide having aspartate transport activity;
A nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1 and encoding a polypeptide having aspartate transport activity;
A nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, And a nucleic acid encoding a polypeptide having aspartate transport activity,
A kit for screening for an activity regulator of an aspartate transport protein comprising a polypeptide encoded by a nucleic acid selected from the group consisting of b) and a lipid.
(Item 22) The kit according to Item 21, further comprising (c) a proton pump.
23. (c) above proton pump is _F o _F 1 -ATPase, The kit of claim 22.
(Item 24) The kit according to item 21, further comprising (d) a membrane potential forming agent.
(Item 25) The kit according to item 24, wherein (d) the membrane potential forming agent is valinomycin.

  In accordance with the present invention, a transporter responsible for aspartate transport and the gene encoding it have been identified. Furthermore, a screening method for a transporter activity regulator using an artificial membrane containing such a transporter is provided. Furthermore, such a screening method also provides a method for screening a candidate substance for a prophylactic / therapeutic agent for a disease involving aspartic acid metabolism and such a candidate substance.

FIG. 1A schematically shows the preparation of reconstituted proteoliposomes using wild-type and mutant sialin. FIG. 1B shows the results of reconstitution of wild-type and mutant sialin and measurement of H ⁺ / sialic acid cotransport activity. Figure _{2, (1) F o F 1} -ATPase only reconstituted liposomes (F-ATP-ase _{liposome), (2)} F o F 1 liposomes were reconstituted -ATPase and sialic (ATP no addition) (- _ATP), a ₍₃₎ F o F 1 -ATPase and reconstituted liposomes (ATP added sialic) (+ ATP) results showing a time course of each L- aspartic acid uptake. FIG. 3 shows aspartic acid transport activity by addition of valinomycin (2 μM), nigericin (2 μM), (NH ₄ ) ₂ SO ₄ (2 mM), CCCP (1 μM), NaN ₃ (1 mM), Evans Blue (1 μM). It is the result of measuring the influence. FIG. 4 shows the results of measuring the effects on the aspartic acid transport activity and sialic acid transport activity due to the addition of various amino acids and other substances. FIG. 5A is a photograph showing that sialin and aspartic acid coexist in the process terminal region of the pineal cell. FIG. 5B shows the results showing that the uptake of aspartic acid decreased when the expression of the sialin gene was suppressed by RNAi. FIG. 6A shows the results of Western blotting with an anti-sialin antibody on the hippocampal P2 membrane vesicle fraction. The arrow is a molecular weight band corresponding to sialin. FIG. 6B shows the results of immunohistochemical analysis of hippocampal slices. In the left diagram of FIG. 6B (“Sialin + Syn”), the signal from the anti-synaptophysin antibody is indicated by an arrow, and the signal from the anti-sialin antibody is indicated by an arrow. In the central view of FIG. 6B (“VGLUT1 + Syn”), the signal from the anti-synaptophysin antibody is indicated by an arrow, and the signal from the anti-VGLUT1 antibody is indicated by an arrow. The right figure of FIG. 6B (“Control + Syn”) shows the results using an anti-synaptophysin antibody (5 nm particles) and control serum (10 nm gold particles). The graph of “glutamate” and “aspartate” of “hippocampus” in FIG. 6C is a result of measuring glutamate uptake activity and aspartate uptake activity of the hippocampal P2 fraction. “Control” is the result using the hippocampal P2 membrane fraction, “+ CCCP” is the result obtained by adding CCCP to the reaction solution used in “Control”, and “tHA” is “Control”. This is a result obtained by adding tHA to the reaction solution used in the above. The graph of “glutamic acid” and “aspartic acid” of “whole brain” in FIG. 6C is a result of measuring glutamic acid uptake activity and aspartic acid uptake activity of the whole brain P2 fraction. “Control” is a result using the whole brain P2 membrane fraction, “+ CCCP” is a result obtained by adding CCCP to the reaction solution used in “Control”, and “tHA” is “ It is the result obtained by adding tHA to the reaction solution used in “Control”. FIG. 7A shows the result of Western blotting with an anti-sialin antibody against pineal gland cultured cells. FIG. 7B shows the result of double staining of cultured pineal cells with anti-aspartic acid antibody (L-Asp), anti-sialin antibody (Sialin), and anti-synaptophysin antibody (Syn). The bar is 10 μm. The result of double staining (Merge) in the upper part of FIG. 7B shows the result of double staining using an anti-aspartate antibody (L-Asp) and the anti-synaptophysin antibody (Syn), and the result of double staining in the lower part of FIG. 7B. (Merge) shows the result of double staining using an anti-sialin antibody (Sialin) and an anti-synaptophysin antibody (Syn). FIG. 7C shows the results of immunohistochemical analysis of cultured pineal cells. In the left diagram of FIG. 7C (“Sialin + Syn”), the signal from the anti-synaptophysin antibody is indicated by an arrow, and the signal from the anti-sialin antibody is indicated by an arrow. In the central view of FIG. 7C (“VGLUT2 + Syn”), the signal from the anti-synaptophysin antibody is indicated by an arrow, and the signal from the anti-VGLUT2 antibody is indicated by an arrow. The right figure of FIG. 7C (“Control + Syn”) is the result using an anti-synaptophysin antibody (5 nm particles) and control serum (10 nm gold particles). Bar indicates 100 nm. FIG. 7D shows the results of specific suppression of sialin expression using siRNA against sialin. “−” Is the result of not adding siRNA, and “+” is the result of adding siRNA. The graph on the left shows the result of dividing the expression level of sialin by the expression level of G3PDH as a relative value with “−” (result of not adding siRNA) as 100%. The right graph shows the result of dividing the expression level of VGLUT2 by the expression level of G3PDH as a relative value with “−” (result of not adding siRNA) as 100%. FIG. 7D is a graph showing the results of aspartic acid release and glutamic acid release for each of the cases where no siRNA was added (“−”) and the case where siRNA was added (“+”). 8 (1) without the addition of _sialic, if ATP was added to the liposomes reconstituted only F _o F 1 -ATPase - the (sialic _{+ ATP), (2) F} o F 1 -ATPase and sialic reconstituted liposomes (ATP no addition) (- _ATP), a ₍₃₎ F o F 1 -ATPase and reconstituted liposomes (ATP added) sialic (+ ATP) results showing a time course of each glutamic acid uptake. FIG. 9 shows the aspartate uptake activity of reconstituted liposomes containing sialin and FoF1-ATPase, in which 1 μM Evans Blue, 1 μM Chicago Sky Blue, and 10 μM DIDS were added, respectively, and preincubated, followed by 2 mM ATP. It is a result. Results were expressed relative to the control. FIG. 10 shows the results of measuring the aspartic acid transport activity using liposomes reconstituted only with sialin without using FoF1-ATPase. “+ Val” is the result when valinomycin is used, and “−Val” is the result when valinomycin is not used. FIG. 11 is a graph showing the aspartic acid, glutamic acid, and sialic acid uptake activities of R39C sialin and H183R sialin.

SEQ ID NO: 1 is the nucleic acid sequence of human sialin, and SEQ ID NO: 2 is the amino acid sequence of human sialin.
SEQ ID NO: 3 is the sequence of the forward primer used for human sialin gene cloning.
SEQ ID NO: 4 is the sequence of the reverse primer used for human sialin gene cloning.
SEQ ID NO: 5 is the sequence of the forward primer used for human sialin gene cloning.
SEQ ID NO: 6 is the sequence of the reverse primer used for human sialin gene cloning.
SEQ ID NO: 7 is the sequence of mouse H183R 1 ^st PCR sense primer.
SEQ ID NO: 8 is the sequence of mouse H183R 1 ^st PCR antisense primer.
SEQ ID NO: 9 is the sequence of mouse H183R 2 ^nd PCR sense primer.
SEQ ID NO: 10 is the sequence of RNAi used for knockdown of the sialin gene.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  The term “transporter” as used herein refers to a substance that transports a substance that cannot permeate the lipid bilayer (eg, aspartic acid) across the lipid bilayer. Typically, the transporter is a membrane protein present in the lipid bilayer. A transporter that is a protein is used herein interchangeably with “transport protein”.

  As used herein, the term “transport activity” refers to the activity of transporting a substance that cannot permeate the lipid bilayer (eg, an anion such as aspartic acid) across the lipid bilayer. The transport activity of aspartic acid is referred to herein as “aspartic acid transport activity”. When using liposomes reconstituted with sialin, the transport activity can be measured using the amount of incorporation into the reconstituted liposome as an index. In addition, the transport activity of sialin in nerve cells can be determined by using the release amount of an object (for example, aspartic acid, glutamic acid, or sialic acid) from the nerve cells as an indicator of the transport activity.

As used herein, the term “proton pump” refers to a protein having transport activity that transports H + using ATP as an energy source. Typical proton pumps include F _o F ₁ -ATPase present in E. coli, mitochondria and chloroplasts, V-ATPase present in vacuoles and chromaffin granules, Na / K-ATPase and H / K present in cell membranes. -ATPase is included, but is not limited thereto.

  The term “artificial membrane” used in the present specification is a membrane artificially prepared from a lipid as a raw material, and is preferably a lipid bilayer membrane, but is not limited thereto. Examples of the “artificial membrane” include, but are not limited to, liposomes.

  As used herein, the term “aspartate transport protein activity modulator” refers to a substance that affects the transport activity of aspartate transport protein. The “aspartate transport protein activity regulator” may be a substance that promotes or inhibits transport activity.

  As used herein, the term “membrane potential forming agent” refers to an agent that forms a membrane potential when added to liposomes. Preferably, the membrane potential forming agent of the present invention is a compound that makes the potential inside the liposome positive. Examples of the membrane potential forming agent include, but are not limited to, valinomycin and bis [(benzo-15-crown-5) -4-methyl] pimelate. When valinomycin is used, it is necessary to form a potassium concentration gradient inside and outside the liposome. When valinomycin is added under conditions where the potassium concentration outside the liposome is high and the potassium concentration inside the liposome is low, a positive potential is formed in the liposome.

As used herein, a “kit” refers to a product comprising a plurality of containers and manufacturer's instructions, and each container comprising a nucleic acid and / or protein of the present invention. If necessary, the kit of the present invention contains an artificial membrane such as a liposome or a lipid for preparing the artificial membrane. If necessary, the kit of the present invention, comprising a proton pump for forming an electrochemical potential of protons in an artificial membrane (ATPase _(e.g., F _o F 1 -ATPase or vacuolar ATPase).

  A “polynucleotide”, “nucleic acid” or “nucleic acid molecule” is a ribonucleotide (adenosine, guanosine, uridine, or cytidine in the form of a phosphate ester polymer that is in single-stranded, double-stranded, or other form; “RNA molecule”) or deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine (DNA molecule)), or any phosphoester analog thereof (eg, phosphorothioate and thioester).

  A “polynucleotide sequence”, “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotide bases (also referred to as “nucleosides”) in a nucleic acid (eg, DNA or RNA) and any chain of two or more nucleotides. Or its complementary strand. Preferred nucleic acids of the present invention include the nucleic acid set forth in SEQ ID NO: 1 and its complementary strands, variants and fragments.

  “Complementary strand” means a strand of nucleotides capable of forming base pairs with a nucleic acid sequence. For example, each strand of double-stranded DNA has a complementary base sequence, and the other strand is a complementary strand when viewed from one strand.

  A “coding sequence” or a sequence that “encodes” an expression product (eg, RNA, polypeptide, protein, or enzyme) is a nucleotide sequence that, when expressed, results in the production of that product.

  A “protein”, “peptide” or “polypeptide” includes a contiguous sequence of two or more amino acids. Preferred peptides of the present invention include the peptide shown in SEQ ID NO: 2, as well as variants and fragments thereof.

  A “protein sequence”, “peptide sequence”, or “polypeptide sequence” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide, or polypeptide.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In the present specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using FASTA, which is a sequence analysis tool, and using default parameters.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. The length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, When it is a nucleic acid, it is substantially separated from a non-nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The terms “purified” and “isolated” as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Means that at least 98% by weight of the same type of biological agent is present.

  In the present specification, “polynucleotide hybridizing under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the hybridizable polynucleotide is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
T _m (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  As used herein, “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can be generated under “highly stringent conditions”. . Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20 % Formamide, 37-50 ° C. As an example, a “moderately stringent” condition of 50 ° C. in 0.015 M sodium ion tolerates a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of a perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. In order to capture more distant related sequences, one of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

  For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is provided by Tm = (2 ° C. for 1 AT base) + (4 ° C. for 1 GC base pair) . The sodium ion concentration in 6 × sodium citrate (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

Natural nucleic acid encoding a protein such as a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a variant or fragment thereof is, for example, a PCR primer and a hybridization probe comprising a part of the nucleic acid sequence of SEQ ID NO: 1 or a variant thereof Is easily separated from a cDNA library having A nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a variant or fragment thereof is essentially 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO ₄ ); 1 mM EDTA; Low stringency conditions defined by hybridization buffer containing 7% SDS at temperature, and wash buffer containing essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); 0.1% SDS at 50 ° C. And more preferably a hybridization buffer comprising 1% bovine serum albumin (BSA) at a temperature of essentially 50 ° C .; 500 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; 7% SDS, and Essentially 50 ° C. 1 × SSC (3 0 mM NaCl; 30 mM sodium citrate); 1% bovine serum albumin (BSA) at low stringency conditions defined by a wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C .; 200 mM phosphorus Sodium acetate (NaPO ₄ ); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS, and essentially 0.5 × SSC at 65 ° C. (150 mM NaCl; 15 mM sodium citrate); 0.1% It can hybridize to the sequence shown in SEQ ID NO: 1 or a part thereof under low stringency conditions defined by a wash buffer containing SDS.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but is not limited thereto. In a particularly preferred embodiment, the Basic Local Alignment Tool (BLAST) known in the prior art (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990). J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Stance and Stance. about). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). about).

  In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used.

  Examples of nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 19 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths, 17 contiguous nucleotide lengths, 18 contiguous nucleotide lengths, 19 contiguous lengths It can be a nucleic acid sequence of nucleotide length, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths, 50 contiguous nucleotide lengths. The nucleic acid sequence used as a primer is a nucleic acid that is at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, most preferably 95% homologous to the sequence described above. Contains an array. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  In order to confirm the presence of the nucleic acid in the present specification, it can be evaluated by any appropriate method including a molecular biological measurement method such as radioactivity method, fluorescence method, Northern blot method, dot blot method, PCR method and the like.

“Antibody” includes, but is not limited to, antibodies and fragments thereof (preferably antigen-binding fragments). The terms include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F (ab) ₂ antibody fragments, Fv antibody fragments (eg, V _H or V _L ), single chain Fv antibody fragments, and dsFv antibodies. Includes fragments. Furthermore, the antibody molecules of the invention can be fully human antibodies, mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, chicken antibodies, humanized antibodies, or chimeric antibodies. The antibody of the present invention specifically reacts with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

  The gene of the present invention can be knocked down (suppressed) using siRNA. A method for preparing siRNA from a predetermined gene is well known. For example, a double-stranded synthetic siRNA annealed by a siRNA provider known in the art (for example, Nippon Easy Tea, Toyama, Japan) is used. It can be obtained. The synthetic siRNA is dissolved in an RNAse-free solution, adjusted to a final concentration of 20 μM, and then introduced into cells. When preparing siRNA, for example, (1) G or C is not continuously present 4 or more, (2) A or T is not continuously 4 or more, (3) G or C is 9 Conditions such as the absence of more than a base may be added. The siRNA of the present invention has 19 base length, 20 base length, 21 base length, 22 base length, 23 base length, 24 base length, 25 base length, 26 base length, 27 base length, 28 base length, 29 base length, Or it is 30 base length. The siRNA of the present invention is preferably 19 bases long. The siRNA of the present invention is also preferably 20 bases long. The siRNA of the present invention is also preferably 21 bases in length. The siRNA of the present invention is also preferably 22 bases in length. The siRNA of the present invention is also preferably 23 bases in length. The siRNA of the present invention is also preferably 24 bases in length.

  The terms “express” and “expression” allow or cause information in a gene, RNA sequence or DNA sequence to become apparent (eg, cells involved in transcription and translation of the corresponding gene) To produce a protein by activating the function). A DNA sequence is expressed in or by a cell to form an “expression product” (eg, RNA (eg, mRNA) or protein). The expression product itself may also be said to be “expressed” by the cell.

  The term “transformation” means the introduction of a nucleic acid into a cell. The introduced gene or sequence may be referred to as a “clone”. A host cell that receives the introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone”. The DNA or RNA introduced into the host cell can be from any source, and can be from a cell of the same genus or species as the host cell or from a cell of a different genus or species.

  The term “vector” refers to a medium (eg, a plasmid) into which a DNA or RNA sequence can be introduced into a host cell so as to transform the host and facilitate expression and / or replication of the introduced sequence as needed. ).

  Vectors that can be used in the present invention include plasmids, viruses, bacteriophages, integratable DNA fragments, and other media that can facilitate the introduction of nucleic acids into the host genome. A plasmid is the most commonly used form of vector, although all other forms of vectors that provide similar functions and are known or known in the art are wild-type herein. Suitable for use. See, for example, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 and Supplements, Elsevier, N .; Y. And Rodriguez et al., (Eds.), Vectors: A Survey of Molecular Cloning Vectors and Their Uses 1988, Buttersworth, Boston, MA.

  The term “expression system” means a host cell and compatible vector that, under appropriate conditions, can express a protein or nucleic acid carried by the vector and introduced into the host cell. Common expression systems include E. coli. E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors.

  Expression of the nucleic acid encoding the polypeptide set forth in SEQ ID NO: 2 of the present invention can be preferably carried out by conventional methods in eukaryotic cells. Suitable host cells for expressing the nucleic acid include higher eukaryotes, animal cells (non-mammalian sources (eg, insect cells)) and mammalian sources (eg, humans, primates, and rodents). 2) established tissue culture cell lines derived from both animal cells).

  Higher eukaryotic tissue culture cells can also be used for recombinant production of the polypeptide set forth in SEQ ID NO: 2 of the present invention. Although any higher eukaryotic tissue culture cell line (including insect baculovirus expression systems) can be used, mammalian cells are preferred. Such transformation, transfection and propagation of cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, J774 cells, Caco2 cells, rat infant kidney (BRK) cell lines, insect cell lines, avian cell lines, and monkey (COS) cells. Strains. Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, an RNA splice site (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors can be plasmids, viruses, or retroviruses that carry a promoter derived from a source such as, for example, adenovirus, SV40, parvovirus, vaccinia virus, or cytomegalovirus. Examples of expression vectors include pCR® 3.1, pCDNA1, pCD (Okayama et al., (1985) Mol. Cell Biol. 5: 1136), pMC1neo Poly-A (Thomas et al., (1987) Cell 51: 503), pREP8, pSVSPORT and derivatives thereof, and baculovirus vectors (eg, pAC373 or pAC610).

The present invention also includes the polypeptide set forth in SEQ ID NO: 2 of the present invention and the polynucleotide set forth in SEQ ID NO: 1 and a second polypeptide portion or a second polynucleotide portion (which may be referred to as a “tag”). , Including fusions. The fusion polypeptide of the present invention can be easily constructed, for example, by inserting the polynucleotide of the present invention or a fragment thereof into an expression vector. The fusions of the invention can include a tag that facilitates purification or detection. Such tags include glutathione-S-transferase (GST), hexahistidine (His6) tag, maltose binding protein (MBP) tag, hemagglutinin (HA) tag, cellulose binding protein (CBP) tag, and myc tag. Is mentioned. Detectable tags (eg, ³² P, ³⁵ S, ³ H, ^99m Tc, ¹²³ I, ¹¹¹ In, ⁶⁸ Ga, ¹⁸ F, ¹²⁵ I, ¹³¹ I, ^{113 m} In, ⁷⁶ Br, ⁶⁷ Ga, ^99m Tc, ¹²³ I, ¹¹¹ In and ⁶⁸ Ga) can also be used to label the polypeptides and polynucleotides of the invention. Methods for constructing and using such fusions are well known in the art.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

(Screening method of activity regulator of aspartate transport protein)
Aspartic acid transfer protein activity modulators can be screened by various methods using the protein of the present invention. For example,
(A) reconstituting ATPase (eg, vacuolar ATPase) and the membrane protein of the present invention into liposomes;
(B) (1) Radiolabeled aspartic acid alone, (2) Radiolabeled aspartic acid and a candidate drug were added to each of the liposomes and incubated,
(C) The liposome is centrifuged to precipitate, and the amount of radiolabeled aspartic acid incorporated into the liposome is compared between (1) and (2).
(D) It is possible to screen for an activity regulator of aspartate transport protein by determining whether the candidate drug has affected aspartate transport activity.

Or
(A) preparing a cell expressing the membrane protein of the present invention (for example, transforming using the gene of the present invention),
(B) The cells were incubated with (1) radiolabeled aspartic acid alone, (2) radiolabeled aspartic acid and a candidate agent,
(C) destroy the cells, prepare the membrane fraction, compare the amount of radiolabeled aspartic acid present in the membrane fraction in the case of (1) and (2),
(D) It is possible to screen for an activity regulator of aspartate transport protein by determining whether the candidate drug has affected aspartate transport activity.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

  According to the knowledge so far, there is no example in which aspartic acid is accumulated alone in synaptic vesicles. It can be found that both aspartic acid and glutamic acid coexist. Therefore, the present inventors hypothesized that the hypothesis contrary to the conventional knowledge (Non-patent Document 3) that the mechanism of concentrating aspartic acid in synaptic vesicles is transported by at least a mechanism different from the glutamate transport mechanism. Based on the hypothesis that the vesicular aspartate transporter VaspT can be thought of as glutamate in addition to aspartate, we attempted to identify the aspartate transporter.

The present inventors established an in vitro measurement method for glutamate transport of VGLUT, and clarified the molecular mechanism of the transport for the first time in the world (J. Biol. Chem., Vol.281, Issue 51, 39499-39506, December). 22, 2006). This method is basically applicable as a function measurement method in a test tube of all transporters. Specifically, an arbitrary transporter is expressed in large quantities in insect cells and purified. In this method, the purified transporter is incorporated into a liposome together with a proton pump purified from Escherichia coli and the transport is measured. This method proved that VGLUT is not only a voltage-dependent glutamate transporter but also a Na ⁺ -dependent phosphate transporter (J. Biol. Chem., Vol. 281, Issue 51, 39499-39506, December22, 2006). This result is groundbreaking in that it shows for the first time that there is at least two independent transport mechanisms in one transporter. This property is called versatility. Based on this discovery, we inferred the properties of virtual VaspT as follows. (1) The fact that it has not been found so far includes candidates for known transporters. (2) Since aspartic acid is an anion, VaspT is a kind of anion transporter. (3) Since glutamic acid should also be transported, it must have properties similar to VGLUT. (4) If the concept of versatility is introduced, other members of the SLC17A family other than VGLUT may be VaspT.

  This time we tested this possibility and found that our expectations were right. That is, it was discovered that sialin which is SLC17A5 among members of SLC17A is VaspT itself. Sialin is known as a lysosomal sialic acid transporter. The sialic acid accumulation disease (sialidosis, infantilelesic acid storage disease, Salla disease) which is a kind of lysosomal disease develops by the functional decline of sialin. In addition to lysosomes, sialin is expressed in many tissues and organelles mainly in the brain (Yarovayaeta1, 2005, J. Neurobiol Dis. 19, 351-365). Therefore, sialin is expected to have physiological functions other than sialic acid transporter. It has been found in the present invention that at least one of its functions is the aspartic acid transport of the present invention. Specifically, the following experiments from Example 1 were conducted.

(Example 1: Reconstitution of sialin)
(PCR)
(Cloning of mouse and human sialin cDNA)
Mouse and human sialin cDNA were obtained by PCR using the following primers.

5'-caccatgaggcccctgcttcggg-3 '(SEQ ID NO: 3) Mouse sialin sense primer
5'-ccacggacacagaaactga-3 '(SEQ ID NO: 4) mouse sialin sense primer
5'-caccatgaggtctccggttcgag-3 '(SEQ ID NO: 5) human sialin sense primer
5'-tcagtgtctgtgtccatggt-3 '(SEQ ID NO: 6) human sialin antisense primer
PCR reaction was performed at 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 45 seconds, 56 ° C for 45 seconds, and 72 ° C for 2 minutes, followed by incubation at 72 ° C for 5 minutes, ExTaqBuffer (Takara), 0.25 mM dNTP mix, 1.6 pmol / μl Primer and 0.5 U Ex Taq (Takara) were added to make a total volume of 16 μl.

  The PCR product was incorporated into the entry vector using the pENTR / D-TOPO cloning kit (No. K2400-20, Invitrogen). The method was performed according to the attached protocol.

(Creation of site-specific mutants)
Primers with the following sequences were used as primers:
5'-agctatgcgggccatgtgg-3 ', (SEQ ID NO: 7) mouse H183R1 ^st PCR sense primer
5'-accacagaggatcatgcataacc-3 '(SEQ ID NO: 8) mouse H183R1 ^st PCR antisense primer
5′-gtaaaacgacggccagtc-3 ′ (SEQ ID NO: 9) Mouse H183R2 ^nd PCR sense primer.

In the 1st PCR reaction, 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 45 seconds, 55 ° C for 45 seconds, 72 ° C for 1 minute, and incubated at 72 ° C for 3 minutes. ExTaq Buffer (Takara), 0.15 mM dNTP mix, 1.6 pmol / μl Primer, and 0.5 U Ex Taq (Takara) were added to the reaction solution to make a total volume of 16 μl.

2 ^nd PCR reaction, after heating of 94 ° C. 2 min, 94 ° C. 45 seconds, 50 ° C. 1 min 30 sec, 72 ° C. 3 min for 35 cycles, and was kept 72 ° C. 5 minutes. The reaction solution was adjusted to a total volume of 16 μl by adding 1 ^st PCR product, ExTaq Buffer (Takara), 0.38 mM dNTP mix, 1.3 pmol / μl Primer, 0.5 U Ex Taq (Takara).

(Link to entry vector)
The PCR fragment was incorporated into an entry vector (pENTR, Invitrogen) using a TOPO cloning kit (Invitrogen). The reaction solution (6 μl) is a solution containing 1 μl of salt solution (Invitrogen), 10 fmol of vector (Invitrogen), and 20 fmol of PCR product. The reaction was carried out at room temperature for 10 minutes, and sialin was incorporated into the entry vector. This was used as a TOPO reaction solution.

(Transformation)
2 μl of the TOPO reaction solution was added to 50 μl of E. coli Mach-1 competent cell (Invitrogen). After leaving on ice for 30 minutes, 250 μl of SOC medium (Invitrogen) was added, reacted at 37 ° C. for 1 hour, and the whole amount was seeded on an LB plate containing 50 μg / ml kanamycin. The plate was cultured overnight at 37 ° C., a single colony was picked up, and cultured overnight in 3 ml of LB medium containing 50 μg / ml kanamycin. A vector containing sialin was obtained from the cultured Escherichia coli using QIAprepSpinMiniprep Kit (Qiagen).

(Example 2: Expression and purification of sialin)
(Recombination into pDEST10)
The sialin cDNA was cloned from the vector prepared in Example 1 into the pDEST10 vector using LR clonase. To 150 mg of the plasmid prepared in Example 1 above, 300 ng of pDEST10 plasmid and 4 μl of LR clonase were added, incubated at 25 ° C. for 1 hour, and then 2 μl of proteinase K was added and incubated at 37 ° C. for 30 minutes. The reaction solution was used to transform DH5α competent E. coli. From the transformed DH5α cells, the plasmid was recovered using QIAprepSpin Miniprep Kit (Qiagen) to obtain pDEST10 / SLC17A5.

(Production of recombinant bacmid)
Using Baculovirus Expression System with Gateway Technology (Invitrogen), the sialin cDNA from pDEST10 / SLC17A5 was integrated into the baculovirus genome (bacmid).

  Specifically, 20 pg of pDEST10 / SLC17A5 was added to 25 μl of DH10Bac competent cells (Invitrogen), left on ice for 30 minutes, and then 225 μl of SOC medium was added at 42 ° C. for 30 seconds. Incubated for 4 hours at 37 ° C., seeded with LB plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline and incubated overnight at 37 ° C. Well, the bacmid was recovered by the miniprep method.

(Miniprep method; for backmid)
The miniprep method used for the production of the recombinant bacmid was performed according to the following procedure. First, DH10Bac having a recombinant bacmid was inoculated into 3 ml of LB medium supplemented with 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline, and cultured at 37 ° C. The cultured E. coli is suspended in 200 μl of solution 1 (50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0), and then 200 μl of solution 2 (0.2 M NaOH, 1% SDS) is added, Mixed. After standing at room temperature for 5 minutes, 200 μl of solution 3 (3M KOAc, 11.5% (v / v) acetic acid) was added and mixed over the counter. After leaving at 4 ° C. for 10 minutes, the mixture was centrifuged (13,000 rpm, 15 minutes, 4 ° C.), and the supernatant was removed. The precipitate was further washed twice with 70% ethanol. TE buffer (10 mM Tris / HCl pH 8.0, 1 mM EDTA) was aseptically added thereto, and stored at 4 ° C.

(Preparation of virus)
The virus used in this example was prepared by the following procedure. First, 9 × 10 ⁵ Sf9 cells were seeded in a 35 mm Petri dish. After exchanging the medium with Grace's Insect Medium (GIBCO) supplemented with 0.35 mg / ml sodium hydrogen carbonate, infection with Sf9 was performed by lipofection using 1 μg of bacmid containing sialin and 6 μl of cellfectin (Invitrogen). I let you. After incubating at 27 ° C. for 5 hours, the medium was replaced with 2 ml of completeTMN-FH, cultured until signs of infection were observed, and the medium was collected. This was designated as P1 virus. Then, 6 × 10 ⁶ Sf9 cells were seeded in a 100 mm Petri dish (50% confluent), 1 ml of a 10-fold diluted virus solution was added, and the mixture was shaken at room temperature for 1 hour. CompleteTMN-FH: 4% Sea Plaque Agarose = 3: 1 After removing the mixed Petri dish medium, this was sealed and cultured at 27 ° C. for 7-10 days with 10 ml of layered agarose. The formed plaque was picked up and infected again, and after 72 hours, this medium was recovered in the same manner as in the case of the P1 virus, and designated as P2 virus.

(Cell recovery and solubilization of membrane fraction)
HighFive cells were infected with P2 virus at MOI = 1 and cultured at 27 ° C. Cells 60 hours after infection were collected with a cell scraper and centrifuged at 700 × g for 10 minutes to remove the supernatant. This was suspended in a disruption buffer (20 mM Tris-HCl pH 8.0, 100 mM potassium acetate, 10% glycerol, 5 mM DTT, 1 μg / ml pepstatin A (Peptide Institute), 1 μg / ml leupeptin (Peptide Institute)). The supernatant was removed again by centrifugation at 700 × g for 10 minutes. This was suspended in a disruption buffer, sonicated (TOMY Ultrasonic Disruptor, Output 4, 30 seconds × 8 times), centrifuged at 700 × g for 10 minutes, and the supernatant was collected. 100,000 × g for 1 hour, 4 hours Ultracentrifugation was performed at 0 ° C., and the resulting precipitate was used as a membrane fraction. To this fraction, solubilization buffer (20 mM MOPS-Tris pH 7.0, 2% octylglucoside (Dojindo), 10% glycerol, 1 μg / ml pepstatin A, 1 μg / ml leupeptin) was added, and a homogenizer was used. The suspension was subjected to centrifugation at 100,000 × g for 30 minutes, and the supernatant was used as a solubilized fraction.

(Purification of sialin using affinity column)
The QIAGENNi-NTA super flow resin was packed in an Econo column (1 mL; 50% slurry), washed with distilled water, and equilibrated with a solubilization buffer having a pH of 8.0. The above-mentioned solubilized fraction was added thereto and adsorbed while stirring at 4 ° C. for 4 hours. This was washed with 15 ml of washing buffer (20 mM MOPS-Tris pH 7.0, 1% octylglucoside, 20% glycerol, 5 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin), and elution buffer (20 mM MOPS- Purified sialin was eluted using Tris pH 7.0, 1% octyl glucoside, 20% glycerol, 60 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin).

(Example _{3: F} o _F 1 -ATPase purification)
Moriyama Y et al. Biol. Chem. 266, 22141-22146 (1991), a proton pump F ₀ F ₁ protein was prepared.

E. coli DK8 containing the large-scale expression plasmid pBWU13 of F ₀ F ₁ was added to Tanaka medium (34 mM monopotassium phosphate, 64 mM dipotassium phosphate, 20 mM ammonium sulfate, 0.3 mM magnesium chloride, 1 μM) containing 0.5% glycerol. After culturing with iron sulfate, 1 μM calcium chloride, 1 μM zinc chloride, 100 μg / ml isoleucine, 100 μg / ml valine, 2 μg / ml thiamine), the cells were collected. All subsequent preparations were performed at 4 ° C.

About 10 g of microbial cells (DK8 / pBWU13) was added to 40 ml of membrane preparation buffer (50 mM at 4 ° C.
Suspended in Tris-HCl (pH 8.0), 2 mM magnesium chloride, 0.5 mM EDTA, 1 mM PMSF, 1 μg / ml leupeptin, 1 μg / ml pepstatin A, 10% (v / v) glycerol, 1 mM DTT), French press Cells were disrupted with (1,500 kg / cm ² ). The crushed liquid was centrifuged at 17,000 × g for 10 minutes, and the obtained supernatant was further centrifuged at 210,000 × g for 1 hour and 20 minutes. The obtained membrane vesicle precipitate was suspended in F ₀ F ₁ preparation buffer (20 mM MOPS / NaOH (pH 7.0), 1 mM magnesium sulfate, 1 mM DTT, 1 mM PMSF, 0.8% octyl glucoside). Centrifuge again. 60 mg of membrane vesicle prepared as a precipitate was suspended in 3 ml of F ₀ F ₁ preparation buffer containing 2% octyl glucoside to solubilize F ₀ F ₁ . The solubilized solution was centrifuged at 260,000 × g for 30 minutes, and F ₀ F ₁ was recovered from the supernatant fraction. The recovered F ₀ F ₁ was purified by 10% (w / v) to 30% (w / v) glycerol density gradient centrifugation (330,000 × g for 5 hours). A glycerol density gradient was made with F ₀ F ₁ preparation buffer containing 1% octyl glucoside. After density gradient centrifugation, the fraction was separated into 10 fractions from the bottom of the centrifuge tube, and the first 4 fractions were collected as F ₀ F ₁ and stored at −80 ° C.

(Example _4: reconstitution into _F o F 1 -ATPase and liposomes of purified sialic)
20 mg of soybean lecithin (SigmatypeIIS) was suspended in a buffer (20 mM MOPS / NaOH pH 7.0, 0.5 mM DTT), and ultrasonically prepared until it became transparent with a bath type ultrasonic device. The prepared liposomes were dispensed and stored at -80 ° C.
Sialic 20μg purified by _F o _F 1 -ATPase _90μg Example 2 was purified by the method of Example 3 was mixed with the liposome 600 [mu] g, and allowed to stand for 15 minutes at -80 ° C., and frozen. This was immediately taken out, rapidly thawed, diluted 20 times with F buffer (20 mM MOPS-Tris pH 7.0, 100 mM potassium acetate, 5 mM magnesium acetate), and centrifuged at 160,000 × g for 60 minutes. To the precipitate, 400 μl of F buffer was added and homogenized to obtain reconstituted proteoliposomes (FIG. 1A).

(Example 5: Sialic acid transport activity in reconstituted proteoliposomes)
(Expression and purification of sialin in insect cells)
Sf9 cells were infected with virus at MOI = 1 for 1 hour, complete TMN-FH medium [Grace's Insect Medium (GIBCO), 10% FBS, 0.35 mg / mL sodium bicarbonate, 4 mg / mL Yeastlate (GIBCO), 3.3 mg / mLlactalbumiin hydrolysate (GIBCO), 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 mg / mL fungizon, pH 6.1], and then cultured at 27 ° C. After 72 hours, the cells were collected with a cell scraper and centrifuged at 700 × g, 10 minutes, 4 ° C. to remove the supernatant. Disruption buffer [20 mM Tris-HCl pH 8.0, 100 mM sodium acetate, 10% glycerol, 0.5 mM DTT, 10 μg / mL pepstatin A (Peptide Institute), 10 μg / mL leupeptin (Peptide Institute)] It was suspended and centrifuged at 700 × g for 10 minutes at 4 ° C. to remove the supernatant. This is suspended again in Disruption buffer, sonicated with TOMY ultrasonicdisruptor (Output4, 30sec × 8 times), centrifuged at 480 × g, 10 minutes, 4 ° C, and the supernatant is recovered. 160,000 × g, 1 hour, After ultracentrifugation at 4 ° C., the resulting precipitate was used as a membrane fraction. Solubilization buffer [20 mM MOPS-Tris pH7.0, 2% octylglucoside (Dojindo Laboratories), 10% glycerol, 10 μg / mL pepstatin A, 10 μg / mL leupeptin] is placed in this membrane fraction and suspended using a homogenizer. 260,000 × g, 30 minutes, 4 ° C. was centrifuged, and the supernatant was used as the solubilized fraction. This solubilized fraction was adsorbed to Ni-NTAsuper flow resin (QIAGEN) at 4 ° C. for 4 hours while stirring. This is washed with 10 mL Wash buffer [20 mM MOPS-Tris pH 7.0, 1% octylglucoside, 20% glycerol, 5 mM imidazole, 10 μg / mL pepstatin A, 10 μg / mL leupeptin], then 2.5 mL Wash Washed with buffer + 500 mM NaCl. It was washed again with 5 mL of Wash buffer, and sialin was eluted with 3 mL of the same solution (Elution buffer) with 60 mM imidazole.

(Measurement of sialin reconstitution and transport activity)
40 μg of purified sialin was mixed with 550 μg of liposomes, allowed to stand at −80 ° C. for 15 minutes, and frozen. It thawed rapidly, diluted 20 times with buffer F [20 mM MOPS-Tris pH 7.0, 100 mM potassium acetate, 5 mM magnesium acetate], and centrifuged at 200,000 × g. BufferF200 μL was added to the precipitate and homogenized to obtain reconstituted proteoliposomes.

  Acidic Reaction Mixture [40 mM MES, 100 mM potassium acetate, 5 mM magnesium acetate, 4 mM potassium chloride] or neutral ReactionMixture [20 mM MOPS / Tris pH7.0, 100 mM potassium acetate, 5 mM magnesium acetate, 4 mM chloride Potassium] is incubated at 27 ° C., and [3 H] -sialic acid (2 μCi) is added to a final concentration of 100 μM. To this was added 1/20 times the amount of reconstituted proteoliposome to initiate the reaction. 120 μL of each sample solution was taken, applied to a Sephadex G-50 fine spin column, and centrifuged at 700 × g, 2 minutes, 4 ° C. The eluate was mixed with 3 mL of clear sol, and the radioactivity contained therein (corresponding to sialic acid incorporated into liposomes) was measured with a liquid scintillation counter (Aloka).

When a pH gradient was artificially applied under the above conditions and the transport of H ⁺ and radioactive sialic acid into the liposome was measured, sialic acid transport activity was shown (FIG. 1B). Transport was dependent on pH and the mutant (H183R) had no transport activity. That is, it was confirmed that the transporter is nothing but sialin.

(Example 6: Aspartic acid transport activity in reconstituted proteoliposomes)
(Demonstration that sialin is VaspT)
40 μg of purified sialin and 90 μg of purified FoF1-ATPase were mixed, and 550 μg of liposomes were mixed therewith, allowed to stand at −80 ° C. for 15 minutes and frozen. It thawed rapidly, diluted 20 times with buffer F, and centrifuged at 200,000 × g for 1 hour at 4 ° C. Buffer F 400 μL was added to the precipitate and homogenized to obtain reconstituted proteoliposomes.

  Reaction Mixture [20 mM MOPS / Tris pH7.0, 100 mM potassium acetate, 5 mM magnesium acetate, 4 mM potassium chloride] was incubated at 27 ° C, 1/10 volume of reconstituted proteoliposome was added, and 2 minutes, Incubated at 27 ° C. One minute after adding ATP to a final concentration of 2 mM, [3H] -aspartic acid (5 μCi) was added to a final concentration of 100 μM to initiate the reaction. 120 μL of each sample solution was taken, applied to a Sephadex G-50 fine spin column, and centrifuged at 700 × g, 2 minutes, 4 ° C. The eluate was mixed with 3 mL of clear sol, and the radioactivity contained therein (corresponding to aspartic acid incorporated into liposomes) was measured with a liquid scintillation counter (Aloka).

(Method of inhibiting aspartate secretion by suppressing sialin expression)
Sialin siRNA TargetSequence: caccagaaactcacaagacaa (SEQ ID NO: 10) Negative control TargetSequence: AllStars Neg. SiRNA (QIAGEN) 3-4 weeks old Wistar rats isolated pineal gland, DMEM medium [DMEM (GIBCO), 6% FBS, 55 μg / L sodium pyruvate, 1.8 mg / mL NaHCO ₃ , 6 mg / mL Glucose, 25 mM HEPES / NaOH (pH 7.4), 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 mg / mL fungizon] . The supernatant was removed by centrifugation at 120 × g for 5 minutes at room temperature. Collagenase (GIBCO) treatment (825 units / mL) was performed at 37 ° C. for 30 minutes. The mixture was centrifuged at 120 × g for 5 minutes at room temperature, the supernatant was removed, PBS (−) was added, and the mixture was centrifuged in the same manner. The supernatant was removed, and trypsin (GIBCO) treatment (0.025%) was performed at 37 ° C. for 20 minutes. Centrifugation was performed at 120 × g for 5 minutes at room temperature, the supernatant was removed, and the medium was added. This centrifugation operation was repeated a total of 3 times. The pineal cells were seeded at 2.5 × 10 ⁵ cells / 3.5 cmdish and cultured at 37 ° C. and 10% CO ₂ . After 24 hours, the medium was changed. After 48 hours, the medium was changed to medium + 10 μM Ara-C. After 96 hours, RNAi treatment was performed. That is, a final concentration of 25 nM siRNA and HiPerfectReagent (QIAGEN) are mixed and allowed to stand at room temperature for 10 minutes. The medium was changed, and the siRNA complex was added, followed by culturing for 72 hours, and the following secretion experiment was performed.

30 minutes in Krebs reaction solution [128 mM NaCl, 1.9 mM KCl, 1.2 mM KH ₂ PO ₄ , 2.4 mM CaCl ₂ , 1.3 mM MgSO ₄ , 26 mM NaHCO ₃ , 10 mM MGlucose, 10 mM HEPES / Tris pH 7.4, 0.2% BSA] Preincubation. Krebs reaction solution or high potassium Krebs reaction solution [75 mM NaCl, 55 mM KCl, 1.2 mM KH ₂ PO ₄ , 2.4 mM CaCl ₂ , 1.3 mM MgSO ₄ , 26 mM NaHCO ₃ , 10 mM Glucose, 10 mM HEPES / TrispH 7.4, 0.2% BSA] was washed once and 1.5 mL of the same solution was added to start the reaction. As needed, 100 μL was sampled and centrifuged (flushed) as a supernatant.

Aspartic acid and glutamic acid were measured by HPLC method. Aspartic acid was measured by HPLC with COSMOSIL 5C ₁₈ -AR II, 4.6 × 150 mm + COSMOSIL5C ¹⁸ -MS II, 4.6 × 150 mm (nacalai tesque) reverse phase column and fluorescence detector L-7480 (HITACHI) . For the injection, the sample and the internal standard Homocysteic acid were derivatized (OPA) containing 29.8 mMo-Phalaldehyde (OPA), 98 mM N-Acetyl-L-Cysteine (NAC), 280 mM sodium borate (pH 9.4), and 30% methanol. -NAC) and allowed to react for 2 minutes. As the mobile phase, solution A [50 mM sodium acetate buffer (pH 5.9)] and solution B [80% methanol (20% solution A)] were used.

The glutamic acid was measured by HPLC equipped with COSMOSIL 5C ₁₈ -AR II, 4.6 mm × 150 mm (nacalai tesque) reverse phase column and fluorescence detector L-7480 (HITACHI). For injection, mix the sample and the internal standard o-phospho-L-serine with derivatization reagent (OPA-MSH) containing 3.73 mMOPA, 18 mM MSH, 106 mM sodium borate (pH 9.4), 73.4% methanol for 2 minutes. It was performed after the reaction. As the mobile phase, solution A [2% tetrahydrofuran, 25 mM sodium phosphate buffer (pH 7.5)] and solution B [65% methanol] were used.

  Under the above conditions, sialin was incorporated into the liposome together with a proton pump, and ATP was added to examine whether or not voltage-dependent aspartate transport occurred. As expected, strong aspartate transport activity was observed (FIG. 2).

Next, when ATP is added under the above reaction conditions, valinomycin (2 μM), nigericin (2 μM), (NH ₄ ) ₂ SO ₄ (2 mM), CCCP (1 μM), NaN ₃ (1 mM), Evans blue ( 1 μM) was added. As a result, it was found that this aspartic acid transport depends on the potential and is not a cotransport with H ⁺ . Further, it was markedly activated by a low concentration of chlorine ions (FIG. 3).

When adding aspartic acid, substances such as various amino acids were added to measure aspartic acid transport activity and sialic acid transport activity. This cis inhibition experiment revealed that sialin transports glutamic acid in addition to aspartic acid, and that the substrate specificity of aspartic acid transport is completely different from H ⁺ sialic acid cotransport. Furthermore, it was actually found that radioactive glutamate was actually transported (FIG. 4).

  The results of repeating the above experiment are shown below:

In the above experiments, 2.9 nmol / min / mg protein for aspartate transport, 2.3 nmol / min / mg protein for glutamate transport, and 67 nmol / min / mg protein for sialic acid transport, 100% (control). ).

(Example 7: Confirmation of aspartate transport activity by aspartate transporter by knockdown) Using a pineal gland for which aspartate secretion has been analyzed, whether or not sialin actually functions as VaspT in the cell is examined. It was. Pineal cells were cultured and the expression of lysialin was suppressed by the RNAi method, and aspartic acid secretion was measured. It was shown that sialin and aspartic acid coexist in the processterminal region of the pineal cell (FIG. 5A).
Specifically, the pineal gland was collected from a 3-week old Wisterrat male, immersed in ice-cooled D-MEM medium (Gibco) containing 6% FBS (Gibco) to remove blood vessels, and then chopped. Centrifugation was performed at 180 × g for 2 minutes, the supernatant was removed, 0.1% collagenase (Gibco) PBS (+) solution was added, and the mixture was incubated for 30 minutes. After centrifugation at 180 × g for 2 minutes, the plate was washed once with PBS, 0.025% trypsin (Gibco) PBS solution was added to the pellet, and incubated at 37 ° C. for 20 minutes. After further centrifugation at 180 × g for 5 minutes and washing with D-MEM medium three times, the same medium was added to 0.5-1 × 10 ⁶ cells / ml. Cells were plated on 18 × 18 mm cover glass (Iwaki Glass) coated with 5 μg / ml polylysine (SIGMA) aqueous solution and cultured in a 10% CO 2 incubator at 37 ° C.

  Pineal cells cultured on a cover glass for 1 week are rinsed 3 times with PBS, the medium is removed, and then PBS (+) solution (pH7.4) containing 4% paraformaldehyde (Nacalai) is added for 20 minutes at room temperature. Fixed with. The cells were washed once with PBS (+), and solubilized by incubation with a PBS (+) solution containing 0.1% TritonX100 for 30 minutes. After washing twice with PBS (+), a PBS solution containing 2% goat serum (Gibco) and 0.5% BSA (SIGMA) was added and blocked at room temperature for 30 minutes. 100 μl of anti-L-Asp, anti-synaptophysin (SY38) antibody or anti-sialin antibody diluted with PBS containing 0.5% BSA (SIGMA) was placed on a cover glass and reacted at room temperature for 1 hour. After the reaction, it was washed 4 times with PBS for 5 minutes, and the secondary antibody (anti-rabbit or mouse IgG-FITC, Amersham) diluted with 0.5% BSA (SIGMA) -PBS was placed on a 100 μl cover glass and allowed to react at room temperature for 1 hour. It was. The plate was washed 5 times for 4 minutes with PBS, a mounting agent (IMMUNON) was placed on the slide glass, and a cover glass was enclosed. The specimen was observed with a confocal laser fluorescence microscope (OLYMPUSFLUOVIEW or OLYMPUSLSM-GB200). The sequences used for RNAi were the sense side r (CCAGAAACUCACAAGACAA) dTdT and the antisense side r (UUGUCUUGUGAGUUUCUGG) dTdG.

  And we found that the secretion of aspartic acid and glutamic acid decreased by RNAi method. From this, it can be concluded that sialin functions as VaspT in the cell (FIG. 5B). By this method, secretion of glutamic acid is also slightly suppressed. Glutamate concentration and secretion in the pineal gland is carried out by VGLUT1 and VGLUT2, but this also indicates that VaspT is also involved, in other words, VaspT also functions as VGLUT. This result shows that aspartic acid transport by sialin actually occurs in vivo.

(Example 8: Screening of activity regulator of aspartate transport activity)
16 μl of proteoliposome prepared in Example 4 and F buffer were added, and incubated in a water bath at 27 ° C. for 2 minutes. Next, a candidate drug is added (about 1 μM to 10 mM drug is added in a volume of about 1/100). Thereafter, ATP is added to a final concentration of 2 mM, and aspartic acid (5 μCi) is added to a final concentration of 100 μM to start the reaction. Each 125 μl of sample solution was collected and applied to a Sephadex G-50 fine spin column. The reaction was stopped by centrifugation at 180 × g for 2 minutes. The eluate is dissolved in 3 ml of clear sol, and the radioactivity contained therein (corresponding to aspartic acid incorporated into liposomes) is measured with a liquid scintillation counter. By comparing the results with the results when no candidate drug is added (eg, the results of Example 6), it can be determined whether this candidate agent promotes aspartic acid transport activity or is inhibited / suppressed. it can.

(Example 9: Localization of sialin in the hippocampus)
When a region in the brain where sialin was present was examined by Western blot using an anti-sialin antibody, it was revealed that sialin was present in the hippocampal P2 membrane vesicle fraction (FIG. 6A). The hippocampal P2 membrane fraction used here was isolated by the following method. The isolated hippocampus was homogenized with 20 mL of SME buffer (0.3 M sucrose, 10 mMOPS / Tris pH 7.0.5 mM EDTA) and centrifuged at 943 × g for 8 minutes. The supernatant was centrifuged again at 17321 × g for 15 minutes to obtain a precipitate. This precipitate was homogenized with 2 mL of the same buffer. This was diluted with 40 mL of 8 mM MOPS / Tris (pH 7.0) buffer and stirred for 30 minutes. This solution was centrifuged at 17321 × g for 15 minutes, and the supernatant was further centrifuged at 200,000 × g for 1 hour. This precipitate was suspended in the same buffer to obtain a P2 fraction.

  The hippocampus was analyzed by immunohistochemistry using an electron microscope. The left figure of FIG. 6B (“Sialin + Syn”) is a result of using an anti-synaptophysin antibody (5 nm particle) and an anti-sialin antibody (10 nm gold particle). Signals from anti-synaptophysin antibodies are indicated by arrows, and signals from anti-sialin antibodies are indicated by arrows. The center diagram of FIG. 6B (“VGLUT1 + Syn”) is the result of using anti-synaptophysin antibody (5 nm particles) and anti-VGLUT1 antibody (10 nm gold particles). Signals from anti-synaptophysin antibodies are indicated by arrows, and signals from anti-VGLUT1 antibodies are indicated by arrows. The right figure of FIG. 6B (“Control + Syn”) shows the results using an anti-synaptophysin antibody (5 nm particles) and control serum (10 nm gold particles). Bar indicates 100 nm. From these results, it was found that sialin was present on vesicles containing synaptophysin (FIG. 6B).

  ATP-dependent glutamate transport has been reported in the hippocampal and whole brain P2 membrane vesicle fractions. 6A and 6B show that sialin having aspartic acid transport activity is present in the hippocampal P2 membrane fraction, so the ATP-dependent aspartate transport activity in the hippocampal P2 membrane fraction was tested. did. “Control” is the result of measuring the aspartic acid uptake activity by the method described in Example 6 using hippocampal P2 membrane fraction (1.5 mg / mL protein). “+ CCCP” is a result of adding 1 μM CCCP to the hippocampal P2 fraction used in “Control” and measuring the aspartate uptake activity by the method described in Example 6. As for “tHA”, 5 mM tHA (D, L-threo β-hydroxyaspartate) was added to the hippocampal P2 fraction used in “Control”, and aspartic acid uptake was carried out by the method described in Example 6. It is the result of measuring activity.

  As a result, ATP-dependent aspartic acid transport activity in the hippocampal P2 membrane fraction was confirmed (“Aspartic acid” in FIG. 6C “Hippocampus”). In addition, as previously reported, the ATP-dependent glutamate transport activity of the hippocampal P2 fraction was also confirmed ("glutamate" in Fig. 6C "Hippocampus").

  When the whole brain P2 fraction was used instead of the hippocampal P2 fraction, ATP-dependent glutamate transport was similar, but ATP-dependent aspartate was lower compared to the hippocampal P2 fraction (Fig. 6C “Whole Brain”).

  This indicates that the aspartic acid transport activity by sialin is higher in hippocampal P2 than in other regions of the brain.

(Example 10: Localization of sialin in the pineal gland)
When the region in the brain where sialin was present was examined by Western blot using an anti-sialin antibody, it was revealed that sialin was present in the pineal fraction (FIG. 7A).

  The pineal cells cultured in the primary culture were double-stained with an anti-aspartate antibody (L-Asp), an anti-sialin antibody (Sialin), and an anti-synaptophysin antibody (Syn). The bar is 10 μm. The result of double staining (Merge) in the upper part of FIG. 7B shows the result of double staining using an anti-aspartate antibody (L-Asp) and the anti-synaptophysin antibody (Syn), and the result of double staining in the lower part of FIG. 7B. (Merge) shows the result of double staining using an anti-sialin antibody (Sialin) and an anti-synaptophysin antibody (Syn). In the result of double staining (Merge), the part indicated by an arrow is a part strongly stained by the two types of antibodies used. This result revealed that sialin co-localized with aspartic acid and synaptophysin.

  The location of sialin and synaptophysin in the pineal gland was analyzed in more detail using a double-labeled immunoelectron microscope. The left diagram of FIG. 7C (“Sialin + Syn”) shows the results using an anti-synaptophysin antibody (5 nm particles) and an anti-sialin antibody (10 nm gold particles). Signals from anti-synaptophysin antibodies are indicated by arrows, and signals from anti-sialin antibodies are indicated by arrows. The center diagram of FIG. 7C (“VGLUT2 + Syn”) is the result of using anti-synaptophysin antibody (5 nm particles) and anti-VGLUT2 antibody (10 nm gold particles). Signals from anti-synaptophysin antibodies are indicated by arrows, and signals from anti-VGLUT2 antibodies are indicated by arrows. The right figure of FIG. 7C (“Control + Syn”) is the result using an anti-synaptophysin antibody (5 nm particles) and control serum (10 nm gold particles). Bar indicates 100 nm. From these results, it was found that sialin was present on vesicles containing synaptophysin (FIG. 7C). Since synaptophysin is a marker of synapse-like microvesicles (SLMV), it was revealed that sialin is localized in synapse-like microvesicles.

  It was confirmed using siRNA that sialin expression in cultured pineal cells was responsible for aspartate transport activity. It was confirmed that the cultured pineal cells specifically suppressed sialin expression using the siRNA of SEQ ID NO: 10 in the same manner as in Example 6 (FIG. 7D). On the other hand, siRNA against sialin did not suppress the expression of VGLUT2, which is a glutamate transporter. Aspartic acid releasing activity from the cultured pineal cells was measured, and aspartic acid releasing activity from the sialin siRNA-treated cells was decreased (left in FIG. 7E). Furthermore, glutamate releasing activity from sialin siRNA-treated cells was also decreased (FIG. 7D “exocytosis” right). From this result, it was confirmed that sialin has aspartic acid transport activity. Furthermore, it was confirmed that sialin also has glutamate transport activity.

(Example 10: Glutamate transport activity by sialin)
In the method described in Example 6, sialin was incorporated into liposomes with a proton pump, and ATP was added to examine whether voltage-dependent glutamate transport occurred. As expected, strong glutamate transport activity was observed (FIG. 8).

(Example 11: Effect of inhibitors on aspartate transport)
Using the same experimental conditions as in Example 6, the inhibitory effect of various inhibitors on aspartic acid transport activity by sialin was determined. Reconstituted liposomes containing sialin and FoF1-ATPase were preincubated with an inhibitor, then 2 mM ATP was added to form a membrane potential difference, and then aspartate transport activity was measured. As a result, aspartic acid transport was inhibited by Evans Blue (1 μM), which is a specific inhibitor of VGLUT. The same diazo dye, Chicago Sky Blue (1 μM) was also inhibited. In addition, DIDS (10 μM) reported to have no effect on sialic acid transport significantly inhibited aspartic acid transport (FIG. 9).

(Example 13: Effect of inhibitors on aspartic acid transport)
In Example 6, the transport activity of aspartic acid was measured by reconstituting sialin and FoF1-ATPase. This reconstitution system is epoch-making in that it is a reconstitution system that can measure the transport activity of aspartic acid that could not be measured conventionally. However, since the reconstitution system contains FoF1-ATPase as a component other than sialin, the possibility of generating noise when measuring the activity of sialin itself cannot be completely denied. Therefore, an aspartic acid transport activity measurement system using liposomes reconstituted only with sialin was constructed without using FoF1-ATPase.

  500 μg of liposomes were mixed with 40 μg of purified sialin, allowed to stand at −80 ° C. for 15 minutes, and frozen. It thawed rapidly, diluted 20 times with sialin reconstitution buffer (20 mM MOPS / Tris, pH 7.0, 100 mM sodium acetate, 5 mM magnesium acetate), and centrifuged at 200,000 × g, 1 hour, 4 ° C. 400 μL of sialin reconstitution buffer was added to the precipitate and homogenized to obtain reconstituted proteoliposomes.

A reaction solution (20 mM MOPS / Tris, pH 7.0, 150 mM potassium acetate, 5 mM magnesium acetate, 4 mM KCl) containing 2 mM valinomycin and [ ³ H] -aspartic acid was added to the reconstituted sialin. Aspartic acid uptake activity was measured (“+ Val” in FIG. 10). As a control, the aspartic acid uptake activity when valinomycin was not added was measured ("-Val" in FIG. 10). As a result, aspartic acid transport activity could be measured by reconstituting only sialin protein without using a proton pump such as FoF1-ATPase as shown in FIG. Although not wishing to be bound by theory, when the potassium concentration in the liposome is low and the potassium concentration outside the liposome is low, the addition of valinomycin transports the valinomycin outside the liposome into the liposome, resulting in a membrane potential of It is considered that sialin transported aspartic acid using the membrane potential as a driving force. This method is an epoch-making method in that the transport activity of sialin itself can be measured.

(Example 14: Transport activity of sialin having Sarah disease (R39C) or ISSD (H183R) mutation)
In patients with Sarah disease, Arg39 of sialin is mutated to Cys (hereinafter referred to as “R39C sialin”). In ISSD (infant-type sialic acid storage disease), His183 of sialin is mutated to Arg (hereinafter referred to as “H183R sialin”). The transport activity of these mutant sialins was tested.

  In the same manner as in the wild-type sialin experiment in Example 6, R39C sialin and H183R sialin were expressed and reconstituted into liposomes, and the aspartic acid, glutamic acid, and sialic acid uptake activities (transport activities) were tested. Membrane potential-dependent aspartate and glutamate transport activities and pH gradient-dependent sialic acid transport activities were shown relative to the activity of wild-type sialin. R39C sialin (Sara disease) lost aspartic acid and glutamate transporting activity, and H183R sialin (ISSD) lost sialic acid transporting activity (FIG. 11).

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  In accordance with the present invention, a transporter responsible for aspartate transport and the gene encoding it have been identified. According to the present invention, a screening method for a transporter activity modulator using an artificial membrane containing such a transporter is provided. Furthermore, such a screening method also provides a method for screening a candidate substance for a prophylactic / therapeutic agent for a disease involving aspartic acid metabolism and such a candidate substance.

Claims (12)

A screening method for an activity regulator of aspartate transport protein,
(A) an artificial membrane,
A nucleic acid that hybridizes under a highly stringent condition with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and that encodes a polypeptide having aspartic acid transport activity;
A nucleic acid comprising a sequence that is at least 90 % identical to the nucleic acid sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide having aspartate transport activity;
A nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, And providing an artificial membrane comprising a nucleic acid-encoded polypeptide selected from the group consisting of nucleic acids encoding polypeptides having aspartate transport activity;
(B) contacting the candidate drug with the artificial membrane;
(C) measuring the aspartic acid transport activity of the artificial membrane; and
(D) determining whether the candidate drug is an activity regulator of aspartate transport protein from the aspartate transport activity measured in step (c);
Including the method. The method of claim 1 , wherein the activity modulator is an inhibitor. The method according to claim 1 , wherein the activity regulator is an activity promoter. The method of claim 1 , wherein the artificial membrane of (a) further comprises a proton pump. The method according to claim 4 , wherein the proton pump is F _o F ₁ -ATPase. The method according to claim 1 , wherein the artificial membrane of (a) further contains a membrane potential forming agent. The method according to claim 6 , wherein the membrane potential forming agent is valinomycin. a) a polypeptide comprising:
A nucleic acid that hybridizes under a highly stringent condition with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide having aspartate transport activity;
A nucleic acid comprising a sequence that is at least 90 % identical to the nucleic acid sequence set forth in SEQ ID NO: 1 and that encodes a polypeptide having aspartate transport activity;
A nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and an amino acid sequence comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, And a nucleic acid encoding a polypeptide having aspartate transport activity,
A kit for screening for an activity regulator of an aspartate transport protein comprising a polypeptide encoded by a nucleic acid selected from the group consisting of b) and a lipid. The kit according to claim 8 , further comprising (c) a proton pump. The kit according to claim 9 , wherein the (c) proton pump is FoF1-ATPase. The kit according to claim 8 , further comprising (d) a membrane potential forming agent. The kit according to claim 11 , wherein the (d) membrane potential forming agent is valinomycin.